It is often advantageous to solubilize polysaccharides in aprotic solvents to carry out reactions that would not be possible in protic media. Such reactions include those which activate polysaccharides using a reagent that is sensitive to water.
Marburg et al., U.S. Pat. No. 4,695,624, describes the functionalization of polysaccharides for conjugation to proteins in order to prepare polysaccharide-protein conjugates, which are useful as vaccines. The polysaccharides must be covalently-modified prior to conjugation by first solubilizing them in aprotic (non-hydroxylic) solvents so that nucleophilic hydroxyl groups of the polysaccharides can react with electrophilic reagents.
Egan, et al. J. Amer. Chem. Soc.. 1986, 108, 5282-5287 describes the adipic acid dihydrazide (AAD) functionalization of bacterial polysaccharide termini with a carbodiimide. A similar reaction with AAD-functionalized proteins would provide polysaccharide-protein conjugates, which also may be useful as vaccines. In order to avoid competing cyclophosphate formation from the O-phosphoryl isourea intermediate, the reaction must be carried out in aprotic media. AAD is a more reactive nucleophile in aprotic solvents than in water, and can effectively compete with intramolecular cyclization.
Previously, the solubilization of polyanionic bacterial polysaccharides in aprotic solvents was achieved by replacement of the alkali metal or alkali-earth metal cations with large hydrophobic cations such as tri- or tetraalkylammonium, 1-azabicyclo-[2.2.2]-octane, and 1,8-diazabicyclo[5.4.0]undec-7-ene. Solubilization was preferably accomplished by passing the polysaccharide through a strong acid cation exchange resin in the tetralkylammonium form.
Solubilizing polysaccharides according to the method described in Marburg et al., and Egan et al., i.e. by passing the polysaccharide through a strong acid cation exchange resin, requires extensive processing time, numerous operations and large quantities of raw material. In addition, yields suffer because of binding of the polysaccharides to the resin.
A purpose of the present invention is to provide an efficient process for solubilizing polysaccharides in aprotic solvents, using precipitation as the means for cation exchange rather than passage through a cation exchange resin. Precipitation increases yield, and reduces processing time, the number of operations and the amount of raw material required to obtain a given amount of solubilized polysaccharide.